Eggs have long been recognized as a source of high-quality protein and other important nutrients in the human diet. Phosvitin is a principal phosphoprotein present in egg yolk and represents about 11% of egg yolk proteins and 4% of yolk dry matter. Phosvitin is derived from the large multidomain vitellogenin precursors, which are synthesized in the liver of vertebrates under stimulation of estrogen and later cleaved into phosvitin, lipovitellin and other proteins (Finn, 2007). Phosvitin contains 12% of nitrogen and 10% of phosphorus, and has a molecular weight of 35 kDa (Mecham and Olcott, 1949; Powrie and Nakai, 1986). Phosvitin contains 217 amino acid residues, of which 123 are serine (Byrne et al., 1984). Of the 123 serine residues, 118 are phosphorylated, making it the most highly phosphorylated protein in nature (Byrne et al., 1984; Clark, 1985; Grogan et al., 1990). Due to the large amount of negatively charged phosphoserine residues, phosvitin exhibits strong metal chelating ability, and is believed to provide metal ions during embryonic development (Taborsky, 1983). Phosvitin exhibits numerous other biological properties including antioxidant and anti-bacterial abilities, and excellent emulsion-stabilizing properties (Albright et al., 1984; Chung and Ferrier, 1992; Nakamura et al., 1998; Sattar Khan et al., 2000).
Phosvitin may exhibit greater metal-chelating ability than casein phosphopeptides which are the phosphorylated fragments derived from bovine milk casein digests and are currently used as calcium supplementing agents. A subunit of casein phosphate has only one to thirteen phosphoserine residues to stabilize amorphous calcium phosphate, whereas a molecule of phosvitin has a greater number of phosphoserine residues, implying higher calcium chelating capacity. Phosvitin may thus serve as a source for the production of bioactive peptides that may improve calcium availability in vivo and increase incorporation of calcium into bone (Choi et al., 2005). Since calcium and phosphorus can be simultaneously supplied, phosvitin peptides may be useful in preventing osteoporosis in women and bone loss in aging population.
However, phosvitin is usually considered nutritionally negative due to its strong affinity to metal ions and resistance to proteolytic actions of proteases (Goulas et al., 1996). 95% of the iron in egg yolk is bound to this protein, but only 30% is biologically available (Greegard et al., 1964; Morris and Greene, 1972). Egg yolk protein and phosvitin may inhibit calcium, magnesium and iron absorptions (Ishikawa, 2007). In contrast, casein phosphopeptides enhance vitamin D independent bone calcification in rachitic children (Mellander and Isaksson, 1950; Mellander, 1950). Phosphoserine residues in casein phosphopeptides play a key role by forming soluble organophosphate salts with calcium to limit its precipitation in the distal ileum (Meisel et al., 2003). The common motif, which consists of three phosphoserine residues and two glutamic acids (Ser(P)-Ser(P)-Ser(P)-Glu-Glu) is widely recognized to contribute to the metal chelating ability of casein phosphopeptides (West and Towers, 1976; Schlimme and Meisel, 1995).
Unlike casein phosphopeptides, common knowledge about phosvitin-derived phosphopeptides is limited due to insufficient hydrolysis. The strong negatively charged side chain of phosvitin hinders the enzymatic access to cleavage sites, so the core of phosvitin remains intact after hydrolysis (Goulas et al., 1996; Khan et al., 1998). Compared with casein, which contains no more than fifteen phosphoserine residues in total and three consecutive phosphoserines in a run (Swaisgood, 2003), phosvitin could be an ideal protein source to produce phosphopeptides, since it contains as many as 123 phosphoserine residues and many of them are in consecutive runs (Grogan et al., 1990).
Phosvitin phosphopeptides exhibit antioxidant activities and calcium-absorption promoting ability in vitro and in situ (Jiang and Mine, 2000; Feng and Mine, 2006; Katayama et al., 2006; Katayama et al., 2007; Xu et al., 2007; Young et al., 2011). However, their application has been hindered by the lack of efficient and economic methods to extract phosvitin from egg yolk and to prepare phosphopeptides.
Phosvitin has been precipitated from egg yolk using magnesium sulfate, followed by ammonium sulfate and ethyl ether extraction (Mecham and Olcot, 1949). The purity of phosvitin (expressed as atomic ratio of nitrogen to phosphorus or “N/P”) was 2.72 with an estimate recovery of 60-70% from egg yolk. Another method involves using butanol to remove lipovitellin, recovering phosvitin by repeated precipitation at pH 1.8, purifying with ether and ammonia extraction, and finally precipitating phosvitin using magnesium sulfate (Sundararajan et al., 1960). The resultant N/P ratio was 2.65. A simplified approach involved diluting egg yolk ten-fold with water to prepare an egg yolk pellet, extracting the lipids in the pellet using hexane/ethanol (v/v, 3/1), and extracting phosvitin with 1.74 M sodium chloride, followed by lyophilization (Losso and Nakai, 1994). The purity of the phosvitin, expressed as N/P ratio, was 3.60. A larger N/P ratio may be related to protein impurities and lower purity. Extraction was improved by conducting 10% sodium chloride extraction and magnesium sulfate precipitation, resulting in a N/P ratio of 3.5 and a yield of 3.3 g per 100 g of dried egg yolk (Castellani et al., 2003). However, such methods are of small scale, inefficient, mediocre in purity, and rely upon organic solvents and non-food compatible chemicals such as magnesium sulfate.
Therefore, there is a need in the art for improved methods of extracting phosvitin, and preparing phosvitin phosphopeptides.